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Phaseolus vulgaris agglutinin detection kit

A common bean lectin and detection kit technology is applied in biological testing, preparation of test samples, measurement devices, etc., and can solve the problems of inability to detect, troublesome to use, and inability to reflect the true content of tetramers, and achieves high accuracy , a strong specific effect

Pending Publication Date: 2020-07-28
JIANGHAN UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

There are two important problems in these analytical methods: first, phase bean lectin is a tetramer formed by PHA-E and PHA-L subunits, and there are mainly five forms: L4, L3E1, L2E2, L1E3, E4, and In the erythrocyte aggregation test, only tetramers containing PHA-E subunits can be detected. L4 does not contain PHA-E subunits that can aggregate erythrocytes, and does not participate in hemagglutination reactions. In addition, blood cell aggregation only has two or more PHA-E Subunits can only be aggregated when combined with red blood cells, and E1L3 tetramers are not sensitive to hemagglutination. Therefore, existing detection methods cannot detect L4 and E1L3 tetramers, and the test results only reflect the contents of E4, E3L1, and E2L2 tetramers. It reflects the real content of L4 and E1L3 tetramers, so it cannot truly reflect the real content of several tetramers; second, the red blood cells in the red blood cell aggregation detection method need fresh rabbit blood each time, and the blood is stored in the refrigerator at most It can only be used for two weeks, so rabbits have to be kept for a long time, and the ruptured red blood cells need to be washed away before each use, and the cell concentration must be adjusted under the microscope, so it is very troublesome to use; third, the red blood cell agglutination test is a non-specific coagulation Therefore, the experiment process will also be interfered by other coagulation substances or anticoagulation substances; fourth, the results of the red blood cell aggregation test in the qualitative experiment with naked eyes are highly subjective, and the methods of spectrophotometer and microplate reader require professional laboratories and Only personnel can operate

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0027] The preparation method of phase bean lectin egg yolk antibody IgY comprises the following steps:

[0028] 1. Preparation of injected samples

[0029] Take 200 μg of the standard phase bean lectin, dissolve it with 200 μL sterile saline, add an equal volume of Freund’s complete adjuvant FCA (the first injection) or Freund’s incomplete adjuvant FICA (the second and third injections), and then use Mix the adjuvant and the immune antigen with the double-push method of the syringe to make it fully mixed into a water-in-oil emulsion, which is used to immunize laying hens, and the injection time is 1, 7, and 14 days respectively.

[0030] 2. Egg collection

[0031] After the laying hens were immunized, eggs were collected 40 days later.

[0032] 3. Crude extraction of egg yolk antibody

[0033] Remove the egg whites from the collected eggs and keep the yolks. Dilute 9 times with distilled water according to the weight of the egg yolk, adjust the pH to 5.2-5.6 with octanoic...

Embodiment 2

[0045] Quantitative detection method of phase bean lectin:

[0046] 1. Pretreatment of kidney bean samples to be tested

[0047] Take 1 g of bean pods or bean seeds, add 1 mL of 0.1M carbonate buffer to grind at room temperature, centrifuge at 12,000×g for 10 minutes, take the supernatant, and centrifuge the supernatant again at 15,000×g for 10 minutes, take the supernatant, and store it at 4°C for later use.

[0048] 2. Incubation of the bean sample solution to be tested

[0049] The kidney bean sample to be tested was diluted 10 times with 0.05M carbonate buffer solution (pH=9.6), and the kidney bean lectin standard was serially diluted with concentrations of 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL mL. Add 100 μL of kidney bean grinding liquid and standard gradient dilution samples to each well of a 96-well polystyrene concave-well plate, and incubate at 37°C for 2 hours.

[0050] Pour off the solution in the 96-well polystyrene concave well plate, add 300 μL of phosphate ...

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Abstract

The invention discloses a phaseolus vulgaris agglutinin detection kit. The phaseolus vulgaris agglutinin detection kit contains a phaseolus vulgaris agglutinin egg yolk antibody; the antibody can specifically recognize two subunits, namely PHA-E and PHA-L. According to the method, the content of the phaseolus vulgaris agglutinin is quantitatively is detected by utilizing an enzyme-linked immunosorbent assay, so that the defects of phaseolus vulgaris agglutinin omitted detection and susceptibility of detection results to blood coagulation or anticoagulation components in an existing method areeliminated.

Description

technical field [0001] The invention belongs to the technical field of detection of plant active components, and in particular relates to a detection kit for quantitative detection of bean lectin. Background technique [0002] Kidney beans are also called green beans, lentils, kidney beans, etc. They are the main vegetables in my country, and they are cultivated in spring and autumn in most areas. Poisoning is caused by eating green beans that have been stored for a long time or improperly cooked, such as cold dishes after blanching, or not cooked enough to fully inactivate lectins during frying. The poisoning incidents mostly occurred in collective canteens. It is caused by phase bean lectin, which is composed of four subunits with a molecular weight of about 30kD, and the molecular weight is about 128KD. The phase bean agglutinin subunits are hemagglutinin (PHA-E) and leukocyte agglutinin (PHA-L). The most important factor of bean poisoning is that the hemagglutinin in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/78G01N27/447G01N1/28G01N33/53G01N33/535
CPCG01N21/31G01N21/78G01N27/447G01N1/286G01N33/53G01N33/535G01N2001/2866Y02A50/30
Inventor 韩雪陈禅友陈高陈泽才余英杨琳
Owner JIANGHAN UNIVERSITY
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